USPTO Patents Application 09998575

A34720-PCT-USA-A

Medicament For topical application

The closure of a wound and the r ;lated hemostasis are effected physiologically by .
extravasating blood coagulating i i the wound bed, thereby causing the closure of small blood
vessels and capillaries. The woui .d healing setting in subsequently is effected by the aid of the
provisional extracellular matrix ( ECM) formed by the coagulated blood (Clark, R.A.F. et al.,
1982, J. Invest. Dermatol. 70:264-269; Clark, R.A.F. (ed.), 1996, The Molecular and Cellular
Biology of Wound Repair, Pleni m Press, New York). That matrix, in addition to blood cells,
essentially consists of fibrin as the structural substance serving as a reservoir for a number of
plasma proteins that are importa it for the beginning of wound healing, such as fibronectin

(Mosesson, M.W. and Umfleet,

R., 1970, J. Biol. Chem. 245:5726-5736; Clark, R.A.F. et al.,

1982, J. Invest. Dermatol. 70:26 4-269), vitronectin (Preissner, K.T. and Jenne, D., 1991,

Thromb. Haemost. 66:189-194)
Sci. 408:595-601), plasminogen

plasminogen (Castellino,F.J. et al., 1983, Ann. NY Acad,
activator (Thorsen, S. et al., 1972, Thromb. Pathol. Haemost.
28:65-74), plasminogen activatcjr inhibitor (Wagner, O.F. et al., 1989, Blood 70:1645-1653),
and alpha2-plasmin inhibitor (Sa^ata, Y. and Aoki, N., 1980, J. Clin. Invest. 65:290-297).

The amount of plasminogen activator, plasminogen activator inhibitor and alpha2-plasmin
inhibitor and their mutual ratios exert an essential control on the subsquent degradation
process of fibrin. Yet, also other substances such as, e.g., thrombin, TGF-beta and PDGF are
contained in that fibrin sceleton, which are necessary for the immigration of cells and the
multiple remodeling (reconstruction) of the provisional ECM to the final ECM comprising the
appropriate cell populations.

In the first place, granulocytes immigrate into the wound area and into the wound closure in
major quantities. They release various substances important for the wound healing procedure
to set in such as, in particular, collagenases and elastases, and extracellularly and
intracellular^ destroy microorganisms that have entered the wound area and are able to
propagate there. While the immigration of granulocytes is nearing its end, monocytes
including macrophages are occurring in the wound area to an elevated extent. On the third
day, fibroblasts will sprout in the wound area, getting as far as to the surface of the wound
closure via fibronectin strands. Followed by the ingrowth of blood capillaries, there will occur *
a number of cell transformations and remodeling processes, which, in most cases, will result
in the complete integrity of the injured tissue (Clark, R.A.F.(ed), 1996, The Molecular and
Cellular biology of Wound Repair, Plenum Press, New York).

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The first attempts to close wounds by the aid of blood plasma, which date back already 80 to
90 years, were not successful because of the relatively low viscosity as compared to blood, the
poor adherence in the wound bed and, in the case of the formation of a clot of coagulated
plasma, the high fragility of such a clot.

The use of enriched fibrinogen solutions instead of plasma for the purpose of stopping
bleeding and closing wounds in the beginning likewise was unsuccessful, but finally a
substantial success could be achieved by raising the fibrinogen concentration of such
fibrinogen-containing solutions to more than ten times the fibrinogen level in plasma
(Loblich, 1975, unpublished communication).

When converting fibrinogen into fibrin, it may happen that the hemostatically effective fibrin
wound closure will be detached by wound bed enzymes after some hours, thereby causing
afterbleeding. The detachment of the fibrin woimd closure from the wound bed is a
substantially more frequent and hence more dangerous procedure than the fibrinolysis of the
whole fibrin woimd closure.

It was proved already by the first successful applications of highly concentrated fibrinogen
solufions (Matras, H. et al., 1972, Wr. Med. Wschr. 122:517-523) and the conversion of
fibrinogen to fibrin by thrombin in the wound area that any detachment of the fibrin wound
closure and the usually involved afterbleedings could be avoided by means of fibrinolysis
inhibitors. Among the low-molecular inhibitors assayed, epsilon-aminocaproic acid and
derivatives could be proved effective, yet they had the disadvantage of rapidly diffunding out
of the coagulated fibrin and of the wound area, and hence losing their topical efficacy.

The admixture of high-molecular inhibitors such as, e.g., aprotinin (Trasylol) was successful •
by causing only a slow difflision of the inhibitor from the wound area, yet with the drawback
that this was a bovine and, thus, xenogenic protease inhibitor likely to induce potential
allergies and anaphylactic reactions. Recently, objections have been raised against the use of
animal material for the parenteral application in men also because of the potential
transmissibility of zoonoses.

WO-A - 99/11301 has proposed to replace aprotinin with elastase inhibitors or other inhibitors
effective against leucocyte proteases. According to the fmdings of the inventor of the present
invention, that proposal involves, however, various disadvantages. It is true that those
inhibitors act directly or indirectly by the inhibition of enzymes capable of activating the
fibrinolytic system, yet they may disturb the setting in of wound healing after granulocyte

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immigration into the wound axea by strongly inhibiting the proteases released by the
granulocytes, such as collagenases and elastases;

Another difficulty in producing pharmaceutical medicaments containing fibrinogen and
thrombin as well as an allogenic protease inhibitor and a transglutaminase zymogen is due to
the virus inactivation of such preparations, which has been required for quite some time. A
large portion of the activity of the protease inhibitor or transglutaminase zymogen
respectively contained in the preparation will be lost by virus inactivation in most cases such
that the preparations obtained after having carried out the virus inactivation process frequently
will exhibit but a low activity of the protease inhibitor or transglutaminase zymogen,
respectively. This may result in an insufficient inhibition of fibrinolytic enzymes present in
the wound bed and, consequently, in the detachment of the fibrin wound closure from the
wound bed.

The present invention has as its object to provide a medicament capable of being applied
topically for the purpose of stopping bleedings and/or closing wounds and/or promoting
v/ound healing, wherein the use of a xenogenic protease inhibitor is to be avoided while,
nevertheless, ensuring to a sufficient extent the inhibition of fibrinolytic enzymes in the
wound bed after application of the medicament such that no detachment of the fibrin wound
closure from the wound bed will occur. Furthermore, any disturbance of the setting in of
wound healing due to an inhibition of the proteases released by the granulocytes immigrated
into the wound area, such as collagenases and elastases, by the medicament is to be largely
avoided.

For a medicament comprising as active substances - produced conventionally of allogenic
plasma or tissue or recombinantly - fibrinogen or fibrin, thrombin and one or several
transglutaminase(s), this object is achieved in that the medicament, as a frirther active
substance, contains one or several protease inhibitor(s) selected from the group consisting of
serpins that do not have inhibiting effects on collagenases and elastases, all of the active
substances being of allogenic origin and having been subjected to a process for virus
depletion and/or virus inactivation, with the proviso that the virus inactivation of the one or
several protease inhibitor(s) has not been carried out in the presence of the other active
substances.

By using as protease inhibitors serpins that do not have inhibiting effects on collagenases and
elastases, the inhibition of the proteases released by the granulocytes immigrated into the

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wound area is largely avoided such that the setting in of wound healing will not be impeded
by the medicament according to the invention.

The present invention is further based on the finding that the inhibitory activity of allogenic
protease inhibitors will be preserved to a substantially better degree if the latter are subjected
to virus inactivation not within a preparation containing one or several of the other active
substances of the medicament, but are virus-inactivated separately from the other active
substances. In this manner, it is feasible to prepare medicaments according to the invention
which contain virus-inactivated allogenic protease inhibitors having sufficient activity so as to
inhibit fibrinolytic enzymes in the wound bed after application of the medicament and
preventing the detachment of the fibrin v/ound closure from the wound bed.

In this context, it is noted that the term "virus inactivation" as used for the purposes of the
present invention does not encompass processes merely aimed at virus depletion.

The medicament according to the invention, i.a., contains fibrinogen or fibrin as an active
substance. This means that, depending on the form of appHcation, either fibrinogen as such or
fibrin formed of fibrinogen by the exposure to thrombin is present in the medicament.

The object of the invention is, thus, a composite medicament which constitutes, or develops, a
sterile, virus-safe, slowly absorbable and remodelable allogenic provisional extracellular
matrix to be applied topically or only forming locally. Since the active substances of these
medicaments are of a high molecular character, thus being potential antigens, only allogenic
active substances - based on the species to which the medicament is to be applied - are used
for the production of these medicaments.

By the addition of flirther allogenic virus-safe active substances during its production or
application, the allogenic provisional extracellular matrix enables the control of the wound
healing, in particular, by said active substances being immobilized to the structural substance
by transglutaminases and being capable of exerting their effects in the immobilized state or
being released during the absorption and remodeling process.

The allogenic transglutaminases contained in the medicament according to the invention, for
instance factor Xllla, cause the allogenic protease inhibitors to be covalently bound to fibrin,
thereby practically preventing any diffusion of the inhibitors fi-om the wound area.

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A preferred embodiment of the medicament according to the invention is characterized in that
all of the active substances are present as an allogenic provisional extracellular matrix in a
single pharmaceutical preparation formulated, for instance, as a gel. The medicament in that
form is directly usable for topical application.

Suitably, the active substances also may be present in two or several separate pharmaceutical
preparations to be mixed prior to or during application, wherein the mixture obtained may be
applied in the liquid form or only after its solidification. The preparations may be available in
liquid form or frozen or freeze-dried for thawing or reconstirution prior to mixing.

The pharmaceutical preparations may be mixed, for instance, at least ten minutes prior to their
application on a wound surface, whereupon the mixture will solidify by forming a slowly
absorbable and remodelable allogenic provisional extracellular matrix, which is applied on the
wound area. It is, however, also feasible to mix the pharmaceutical preparations and apply the
liquid mixture topically immediately thereupon, for instance in the form of a spray, so that a
slowly absorbable, remodelable allogenic provisional extracellular matrix will form only
locally.

It is preferred that the concentration of fibrinogen and protease inhibitors in the medicament
be chosen such that the liquid mixture contains at least 30 g/1 of fibrinogen and 500 arbitrary
plasma units of protease inhibitors/1.

Preferably, fibrinogen and thrombin are each present in separate pharmaceutical preparations,
the remaining active substances, independent of one another, being contained in one or both
of said preparations and/or in a further preparation.

By appropriately composing the thrombin-containing pharmaceutical preparation, the
invention enables substantial quanrities of thrombin to be still generated in the formed fibrin
even some hours after mixing with the fibrinogen-containing solution and solidification of the
mixture. This is of great importance both to the stability and to the quality of the allogenic
provisional extracellular matrix thus formed.

The pharmaceutical preparations contained in the medicaments according to the invention
advantageously also may be applied on allogenic or biocompatible carrier materials subjected
to a process for virus depletion and/or virus inactivation. Depending on the purpose of
application, the carrier materials may have different forms. The preparation of a ready-made

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medicament may, for instance, be effected by applying the yet liquid mixture of the
pharmaceutical preparations onto the carrier material.

A preferred embodiment of the medicament according to the invention is characterized in that
it contains, as further active substances, allogenic coUagens subjected to a process for virus
depletion and/or virus inactivation. The additional use of coUagens in the preparation or
formation of the allogenic provisional extracellular matrix consideraby enhances its
biomechanical quality.

Suitably, the medicament according to the invention contains one or several further allogenic
active substance(s) subjected to a process for virus depletion and/or virus inactivation and
selected from the group consisting of fibronectin, vitronectin, thrombospondin, tenascin,
laminin and proteoglycans. By adding substances such as, e.g., vitronectin, further
enhancement of the effect of certain protease inhibitors may be achieved.

It is, moreover, advantageous if the medicament according to the invention contains one or
several further allogenic active substances subjected to a process for virus depletion and/or
virus inactivation and selected from the group consisting of growth factors, chemotactic
substances, cell stimulating and/or proliferation enhancing enzymes and enzyme inhibitors,
proliferation inhibiting eiizymes and enzyme inhibitors, cytokines and particulately formed
cell elements. The application of allogenic enzyme inhibitors in the context of wound closure
offers the opportunity to obtain permanent healing, in particular, in the case of skin ulcera.

Preferably, the medicament according to the invention contains additions of allogenic
plasmatic enzymes, or enzymes obtained from tissues, zymogens and/or enzyme inhibitors
subjected to a process for virus depletion and/or virus inactivation.

Depending on the clinical requirement, it is suitable if the medicament according to the
invention is supplemented with antiadherent, antiphlogistic, antimicrobial and/or cytostatic
agents subjected to a process for virus depletion and/or virus inactivation, if necessary. The
addition of antiphlogistic and antiadherent allogenic agents such as, e.g., certain
immunoglobulins or other active substances occurring in plasma and having such effects
renders feasible the application of an allogenic provisional extracellular matrix even where
postoperative adhesions must be feared.

The allogenic provisional extracellular matrix forming in the appHcation of the medicament or
being present as such may function as a reservoir or depot for substances that are to be slowly

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released from the matrix. A preferred embodiment of the medicament according to the
invention, therefore, is characterized in that it contains additions of active substances that are
to be absorbed slowly and, if necessary, have been subjected to a process for virus depletion
and/or virus inactivation.

Advantageously, allogenic zymogens and/or enzymes of the coagulation cascade, which have
been subjected to a process for virus depletion and/or virus inactivation are additionally
contained in the thrombin-containing pharmaceutical preparation of the medicament
according to the invention.

Preferably, the medicament according to the invention additionally contains allogenic
particulate cell elements, cells and/or tissues, which may be applied on virus-safe
microspheres consisting of allogenic fibrin and/or allogenic collagen and/or allogenic
collagen plus allogenic fibrin.

A drawback of the hitherto known fibrinogen- or fibrin-containing pharmaceutical
preparations is the relatively high fragility of the fibrin formed, even if very high percentage
fibrinogen solutions are used.

Although the fibrin forming during coagulation is crosslinked to form a sceleton on account
of the presence of factor Xllla - a transglutaminase forming in the pharmaceutical preparation
of factor Xni contained in the same by thrombin action -, yet high thrombin and factor XHI
concentrations must still be contained, or form, in the fibrin in order to ensure as complete a
reaction as possible to occur between the crosslinking sites within the fibrin. Even such fibrins
are, however, difficult to use because of the persistently present fragility and the locally
formed fibrins likewise continue to exhibit poor mechanical strengths.

A preferred embodiment of the medicament according to the invention, therefore, is
characterized in that the allogenic provisional extracellular matrix is solidified by the
application of pressure and/or by means of dehydrating agents. In that case, the matrix may be
treated with allogenic transglutaminases prior to, during and/or after solidification by
dehydration.

Such allogenic matrices, due to their sufficient mechanical stability, may also be used as
artificial allogenic skins. It has, thus, also become feasible to produce a skin replacement
exclusively of human substances and additionally introduce in such a preparation also
particulate cell elements, cells and tissues, thus offering new opportunities of medical use.

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It is also feasible by means of the pharmaceutical preparations according to the invention to
fill up body sites afflicted by a strong degradation of endogenous tissue so as to obtain long-
term healing.

Concentrated fibrinogen solutions involve a number of drawbacks. They offer reduced storage
stability and must be deepfrozen or freeze-dried for storage and cannot always be made usable
or reconstituted in a satisfactory manner by thawing or redissolving. Moreover, the
dissolution of a fibrinogen lyophilisate requires some time. Solubilizers or readily soluble
fibrinogens, in most cases, are cytotoxic and, therefore, not suitable for an undisturbed wound
healing.

The greatest difficulty in the storage of fibrinogen-containing products is the instability of
fibrinogen, since traces of impurities'of coagulation factors will cause the slow conversion of
fibrinogen to insoluble fibrin, which no longer will allow the application of such a
pharmaceutical preparation.

The present invention, consequently, also relates to a process for preparing a fibrinogen-
containing solution which, as such or as a component of the medicaments according to the
invention, is storable at refrigerator temperature or room temperature, wherein the fibrinogen
solution or a fibrinogen-fibronectin solution is prepared of recombinantly produced fibrinogen
or of fibrinogen obtained from plasma by fractionation with glycine at temperatures of below
0°C.

Fibrinogen-containing solutions prepared in accordance with the invention are stable and may
be stored in the liquid state with or without stabilizers for more than two years at a
temperature of 4°-8°C or in a frozen or lyophiUzed state without insoluble fibrin precipitating
or fibrinogen cleavage products forming due to the formation of plasmin during storage to an
extent disturbing coagulability. Such solutions will coagulate neither after the addition of
thromboplastin and taipan viper venom nor by the addition of activated partial thrombo-
plastin. No coagulation procedures will be induced by storage in the deepfrozen state and after
thawing of the fibrinogen-containing solutions according to the invention, and the solutions
obtained will be stable for at least some hours. LyophiUzed preparations are readily and
completely reconstimtable by appropriate solvents.

The present invention also provides for a medicament containing a highly purified fibrinogen-
containing or fibrinogen-fibronectin-containing preparation whose aPTT and taipan viper

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venom prothrombin time at 37°C are no less than 200 or 300 seconds, respectively, and which
has a stability enabling it to be stored for more than two years at a temperature of 4°-S°C in
the liquid state with or without stabilizers as a component of medicaments or as such or in a
frozen or lyophilized state, without causing insoluble fibrin to precipitate or fibrinogen
cleavage products to form due to the formation of plasmin during storage.

Despite the use of exclusively allogenic active substances, neoantigens may form in the virus
inactivation of medicaments composed of allogenic active substances, either by the
interaction of two or several active substances of the medicament during virus inactivation
and/or by the interaction with impurities still contained in the active substances.

Another drawback in the virus inactivation of mixtures of active substances resides in the
frequently strongly different inactivation of the individual active substances themselves,
thereby rendering difficuU or impossible the formulation of the respective medicaments.

The invention, therefore, also relates to a process for obtaining pathogen-free active
substances contained in the medicaments according to the invention, comprising a
combination of depletion processes and inactivation processes including steps of virus
depletion and virus inactivation, wherein ulfracentrifugations and ultrafiltrations including
nanofiltrations and/or adsorptions of pathogens at temperatures of below 0°C are used for
virus depletion and at least two different virus inactivation processes are carried out, in which
heat pulse processes of below 3 seconds and/or intensive, laser pulse radiation with or without
photodynamic substances and/or detergents are used along with hydrophobic wetting agents.

The process according to the invention enables the virus inactivation of active substances
having sufficient degrees of purity prior to their formulation, whereby it is ensured by an
appropriate choice of the inactivation processes that all of the active substances are equally
vims inactivated without excessive losses. In that manner, the induction or triggering of
allergies, anaphylactic reactions and autoimmune processes by medicaments containing such
active substances may be avoided to the major extent.

The process according to the invention, in particular, also enables a particularly gentle virus
inactivation of the protease inhibitors contained in the medicaments according to the
invention.

The invention, furthermore, relates to a process for covalently binding to a biological matrix
active substances contained in the medicaments according to the invention, wherein high

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concentrations of allogenic virus-depleted and/or virus-inactivated transglutaminases, which
exceed the 10 time concentration range of the activated concentration of the factor Xllla
zymogen occurring in plasma, are used for the enzymatic catalysis of the binding reaction.

Due to a sufficiently high and sustained thrombin concentration in the allogenic provisional
extracellular matrix and with a sufficient quantity of factor XIII being present, a sufficient
quantity of factor Xllla can be formed, which, on the one hand, causes crosslinking of the
basic substance(s) fibrin with or without collagen and, on the other hand, generates TAFI in
the presence of the TAFI zymogen. TAFI cleaves the plasmin receptor from the fibrin,
thereby providing a high stability of the fibrin-containing allogenic provisional extracellular
matrix against fibrinolytic enzymes. Furthermore, it is feasible according to the invention, due
to a high and sustained transglutaminase concentration in the allogenic provisional
extracellular matrix, to covalently bind appropriately virus-inactivated allogenic protease
inhibitors such as alpha2-antiplasmin, PAI-1 and others, thus preventing them from diffusing
out of the allogenic provisional extracellular matrix. The same holds for other active
substances having protein character and capable of being covalently bound to the sceleton
substance by the aid of transglutaminases.

The invention, furthermore, relates to a process for preparing a fibrin-containing low-water
gel having a water content of between 20 and 90 %, wherein water is removed from the
fibrin-containing gel by applying atoxic, pharmaceutically usable dehydrating agents, in
particular polyethylene glycol, or by introducing the gel into such agents, wherein the gel may
be treated with transglutaminases before or after dehydration.

The producfion of a fibrin-containing gel poor in water, having a water content of between 20
and 90 %, in accordance with the invention also may be effected by a process in which water
is removed from the fibrin-containing gel by applying high pressures, said pressures being
gradually increased in order to avoid destruction of the gel. The gel may be treated with
transglutaminases before, during and/or after the application of pressure.

The invention also relates to a process for solidifying a fibrin-containing gel with and without
dehydration, characterized in that the fibrin-containing gel is placed in one or several metallic
ion-containing solution(s), in particular solutions containing zinc and aluminum ions in
concentrations ranging from 0.01 to 2 molar.

Furthermore, the present invention provides a lyophilized fibrin-containing gel prepared by
the addition of plasticizers, in particular glycol, prior to its solidification and lyophilization.

A34720-PCT-USA-A

The aforementioned processes according to the invention for preparing fibrin-containing low-
water gels as well as solidiiying fibrin-containing gels with a particular advantage may be
used for producing medicaments according to the invention.

A disadvantage of the hitherto used fibrinogen-containing preparations in the topical
application of fibrinogen-containing mixtures of active substances is their too low viscosity
prior to solidification. As a result, the applied mixture may readily and quickly run off the site
of application, which calls for a higher consumption of the medicament, on the one hand, and
leads to an unsatisfactory appHcation, in particular in connection with surgical interventions
where an application is required in the operation area, on the other hand.

The invention, therefore, also relates to a process for preparing a highly viscous fibrinogen-
containing solution, wherein a sterile, virus-depleted and/or virus-inactivated fibrinogen
solution per gram of fibrinogen is mixed slowly and under vigorous stirring and under sterile
conditions with a one hundredth to one tenth imit of sterile, virus-depleted and/or virus-
inactivated thrombin dissolved in a volume as small as possible. The highly viscous
fibrinogen-containing solutions prepared by the process according to the invention with
particular advantage may be used for preparing medicaments according to the invention.

The present invention, furthermore, provides a process for determining the adherence of a
fibrin clot in the wound bed, in which fibrin clots at increasing amounts of protease inhibitors
are formed on the surfaces of suitable tissue cultures, in particular human fibroblasts, with a
small addition of one or several staining substances and/or one or several water-insoluble
substances yielding a stained or opalescent fibrin clot, and the tissue cultures are kept in
motion by slight shaking or rotating while determining the time after which the fibrin clots
detach fi-om the surface of the tissue culture.

The process according to the invention enables the determination of those amounts of protease
inhibitors which are required in an allogenic provisional extracellular matrix as, for instance,
present in, or formed by, the medicaments according to the invention in order to leave such a
matrix on the site of application for at least several days and, thus, prevent its detachment
from the wound bed as well as afterbleeding caused thereby.

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Examples
Example I :

Preparation of a stable fibrinogen solution.

100 1 plasma suitable for the preparation of medicaments, or the Cohn fraction I obtained
therefrom by means of a cold ethanol precipitation, or cryoprecipitate obtained by freezing the
plasma and careful thawing are used as a starting material.

The plasma as such, the Cohn firaction I and the cryoprecipitate after dissolution in about 20
hters of 0.9 % NaCl and 0. 1 % sodium citrate buffer pH 7, are supplemented to saturation
with solid glycine having a degree of purity suitable for pharmaceutical preparations while
being cooled to -2°C to -3°C, stored at that temperature for a minimum of 10 to a maximum
of 15 hours, separated from undissolved glycine, and the fibrinogen- fibronectin-containing
precipitate is separated by centrifugation in a high-speed centrifuge.

The. superaatants may be used for further processing in order to isolate other plasma proteins
therefi-om.

The gelatinous precipitate is removed from the rotor of the centrifuge, dissolved and
precipitated with glycine as before. The sediment obtained after centrifugation is dissolved in
0. 1 % citrate and this procedure of reprecipitation is repeated until a sample of the obtained
sediment taken up in distilled water at a fibrinogen content of 0.1 % to 0.2 % at 37°C with a
PTT reagent yields an aPTT of no less than 200 s. Likewise, a coagulation time of no less
than 300 s must be reached after the addition of thromboplastin and taipan viper venom.

The redissolved final precipitate suitable for farther processing is largely freed of glycine by
diafiltration against an 0.1 % citrate solution and thereby concentrated to a protein content of
from 2 % to 3 %.

The thus obtained fibrinogen still contains a substantial amount of fibronectin. If desired,
fibronectin may be separated by precipitation with 17 % glycine at -2°C to -3°C. In doing so,
practically all of the fibrinogen is precipitated and fibronectin with small amounts of
fibrinogen is contained in the supernatant. By saturation with glycine, the two proteins may be
precipitated and recovered together. Possibly present low activities of plasminogen activator

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or plasmin may be rendered ineffective by the addition of small quantities of low-molecular
inhibitors such as epsilon-aminocaproic acid or derivatives.

The virus inactivation of the highly purified fibrinogen or of the fibrinogen-fibronectin
complex may be effected by admixing the plasma of the dissolved Cohn fraction I or of the
dissolved cryoprecipitate with detergents and wetting agents or by way of a heat pulse or laser
light process. The virus-inactivated fibrinogen solution may be stored as such at a refrigerator
temperature at 4°C to 8°C, or may be deepfirozen or lyophilized for storage.

Example 2:

Preparation of a thrombin solution having thrombin-generating potential

This thrombin solution is obtained by mixing equivalent amounts of two solutions obtained
from human plasma suitable for the production of medicaments for use in humans. The two
solutions are free of pyrogen, free of Ca ions and sterile. One solution contains virus-safe
thrombin in a concentration to be selected from 20 to 2,500 units/ml, the other solution
contains a virus-safe prothrombin coagulation factor mixture capable of generating at least
1,000 thrombin units per ml after the addition of Ca ions.

To that mixture is admixed a one hundredth volume part of a sterile solution of 10 %
polyethylene glycol (pharmaceutical purity) and, depending on the dosage desired, the
mixture is then filled into vials or ready-made syringes and deepfrozen, optionally freeze-
dried, sealed, stored, labeled and packed.

Example 3:

Production of medicaments for topical application and suitable for use in humans, from fibrin-
containing and thrombin-containing pharmaceutical preparations.

800 ml of a pyrogen- free liquid active substance preparation according to Example 1 obtained
from human plasma suitable for the production of medicaments for use in humans and having
a fibrinogen content of at least 6 % are used to dissolve a freeze-dried, sterile, pyrogen- free
virus-inactivated pharmaceutical preparation obtained from plasma suitable for the production
of medicaments for use in humans and containing 1,200 arbifrary units, based on human
plasma, of PAJ-1 or any other serpin or serpin mixture that does not exhibit a coUagenase- or

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elastas.e-inhibiting effect and a transglutaminase zymogen content equivalent to at least 4,000
units of factor XIII. In addition, the preparation contains 2 g CaCh-

This fibrinogen-containing pharmaceutical preparation is then mixed with the thrombin-
containing preparation indicated in Example 2 after reconstitution of the same with a suitable
amount of water for injection, i.e., 4 parts of the fibrinogen-containing preparation are mixed
with 1 part of the thrombin-containing preparation.

Example 4:

Preparation of an allogenic provisional extracellular matrix as a medicament.

A defined amount of a mixmre according to Example 3 is poured into a desired sterilized
mold and incubated for 5 hours under sterile conditions at between 35°C and 37°C. The sterile
pyrogen-free, virus-safe extracellular matrix formed in the mold, which is allogenic for use in
men, is packed into sterile water-vapor-tight polyethylene sheaths, sealed and stored at
refiigerator temperature at 4°C to 8°C to be released according to the respective quality
controls. After labeling, packing and release, the medicament may be put on the market.

Example 5:

Preparation of an allogenic provisional extracellular matrix by mixing three pharmaceutical
preparations offered as a composite medicament.

The medicament contains three pharmaceutical preparations which, after mixing, form an
allogenic provisional extracellular matrix:

a. a 6 % fibrinogen solution according to Example 3,

b. a freeze-dried mixture of transglutaminase zymogen, serpin or serpin mixture, free of
coUagenase and elastase inhibiting effects and calcium chloride in the amounts indicated in
Example 3

c. a freeze-dried thrombin solution according to Example 2.

In addition, the medicament contains water for injection, which is used in order to dissolve the
thrombin-containing pharmaceutical preparation according to Example 2, while the
pharmaceutical preparation containing the transglutaminase zymogen and the seipin is
dissolved in the 6 % fibrinogen solution according to Example 3. After mixing of the

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fibrinogen-containing solution with that containing thrombin and prior to the coagulation of
that mixture, the latter either is introduced into a desired sterile pyrogen-free mold and
removed from the mold after solidification and applied topically or the still Hquid mixture is
applied topically to the desired site or sites with a provisional extracellular matrix allogenic to
man locally forming only then.

Example 6:

Biomechanically reinforced allogenic provisional extracellular matrix using allogenic
collagen.

Into the mixture of the pharmaceutical preparations according to Examples 4 and 5 may also
be introduced fibrillary sterile, pyrogen-free, virus-safe human collagen of between 5 and 100
mg/ml, or the mixture is applied on a rapidly absorbing foamed collagen fleece with the
thrombin content of the mixture being adjusted such that fibrin formation occurs only after the
end of the absorption procedure. Such a fleece may be used immediately after the setting in of
the coagulation procedure or worked up to a ready-made medicament packed under sterile
conditions after a 5 hour incubation at between 35°C and 37°C.

Example 7:

Determination of the required concentrations of non-collagenase-inhibiting or non-elastase-
inhibiting serpins.

A selected fibroblast cell line whose selection has been effected in terms of maximum
fibrinolytic activity is introduced at a cellular density of about 10^ cells per ml into tissue
culture flasks each having a growth area of at least 30 cm^ in such an amount that 10^ cells are
present per cm" tissue culture area. After the addition of at least 10 ml tissue culture medium,
the flasks are incubated until a complete cell lawn has formed. If required, a change of
medium is effected at predetermined intervals.

After a complete cell lawn has grown, the medium is removed and the cell lawn is washed
twice with at least 20 ml medium each, the medium is removed from the flask to the major
extent and 50 fil of a mixture set out below are each applied, by means of suitable pipettes,
onto the exactly horizontally put cell lawn on sites marked on the external sides of the flasks
and, after this, the tissue culture flasks are closed and stored for 1 hour at room temperature,
then are again filled with medium as before, closed and kept in an incubator at 37°C on a

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tissue culture rocker at a rocking rate of about 10 tilts per min and then inspected for the
detachment of the fibrin-containing clots applied on the cell lawn after 30, ICQ and 300
minutes and, thereafter, every 8 hours.

The application of the mixtures indicated below is effected in the following meinner:

Plasma supplemented prior to mixing with methylene blue to the desired extent and one
thousand units of thrombin/ml plasma is applied onto six previously marked sites. By way of
experiment, mixtures according to Example 3 are prepared, which are stained with a desired
amount of phenolic red and contain different amounts of plasmatic serpins or recombinantly
produced serpins. Of each serpin concentration, 6 x 50 )il of the mixture are likewise applied
on the marked sites. After solidification .of the mixture samples apphed, it is proceeded as
indicated above.

The points of time at which the fibrin-containing samples detach ft-om the tissue culture
surface are then determined. The detachment times of the serpinrcontaining samples are
compared to those of the plasma samples. Advantageously, those serpin concentrations are
chosen, which induce detachment times at least equal to those observed with the plasma-
produced clots applied.